Microorganisms primarily acquire phosphorus (P) in the form of inorganic phosphate (PO4-3 or Pi) through expression of a suite of phosphate scavenging or phosphate transporter systems in response to limiting environmental phosphate. One such system is the Pit family of single protein Pi transport systems found in all domains. These vary in size from 300 to 800 amino acids (a.a.) in size.
Previously, the pit gene of the soil bacterium Sinorhizobium meliloti, was found to encode a 334 a.a. Pi uptake system (KM 1-2µM) that is repressed in low Pi conditions. However, the S. meliloti pit gene is encoded in an operon and overlaps the coding sequence of a protein of unknown function, which was denoted as pap (pit-accessory protein).
Using a conditional Pi-transport deficient mutant strain of S. meliloti, the effects of pap or pit mutations on Pit-mediated Pi uptake were studied by conducting growth experiments in minimal media (with Pi as the sole source of P) and Pi uptake experiments. Both pap and pit deletions resulted in a loss of growth and Pi uptake, which could be complemented by integration of the pap and pit genes into the deletion locus.
Heterologous Pap-Pit systems from Bacteroides thetaiotaomicron and Shewanella oneidensis were found to have KM values (17 and 8.5 µM, respectively) similar to previously reported values of S. meliloti Pap-Pit. However, the Shewanella Pit protein was capable of transporting Pi in the absence of the cognate Pap protein, albeit with greatly reduced velocity at all measured concentrations.
Pap-Pit orthologs were identified in ~2000 diverse prokaryotic proteomes using Pfam motifs of Pit (PHO4) and Pap (PhoU_div) protein domains. pap-pit operons were found in a third of all proteomes, and were predicted to be a co-transcribed operon in >95% of cases. This provided additional evidence that Pap is directly involved in Pit-mediated Pi uptake, and also that Pap-Pit systems have a significant role in microbial Pi uptake.
Pap protein sequences and structures show striking similarities with that of PhoU, a protein of unknown function implicated as a modulator of the Pst uptake system. Pap and PhoU proteins share highly conserved putative metal-binding motifs (E/DXXXD) of which several Pap missense mutations were found to result in reduced Pi transport. This suggests that like PhoU, Pap may function as a modulator of Pi uptake by an interaction with its cognate transporter, Pit. However, the molecular mechanisms of PhoU and Pap proteins have yet to be defined. / Thesis / Master of Science (MSc) / Microbes acquire and assimilate phosphorus (P) in the form of inorganic phosphate (Pi) through a variety of mechanisms. Pit (Pi transporter) are a family of diverse transporters found in all kingdoms of life. Unlike other Pit systems, the Sinorhizobium meliloti pit gene is encoded in an operon with a protein of unknown function, denoted the pit-accessory protein (pap). Using S. meliloti Pap-Pit and orthologues from other bacteria as model systems, we demonstrate that Pap functions as a positive modulator of Pi uptake via Pit, as Pap is required for active uptake of Pi. Pap-Pit systems are found in 30% of all bacteria and archaea, and thus broadly distributed. Understanding the mechanism of Pap-Pit has biotechnological applications, as multiple Pap-Pit systems are present in phosphorus-accumulating bacteria utilized for waste-water treatment.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22200 |
| Date | January 2017 |
| Creators | Hsieh, Daniel Hsieh |
| Contributors | Finan, Turlough, Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
Page generated in 0.0023 seconds